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Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

Wartenberg A, Linde J, Martin R, Schreiner M, Horn F, Jacobsen ID, Je S, Wolf T, Kuchler K, Guthke R, Kurzai O, Forche A, d'Enfert C, Brunke S, Hube B - PLoS Genet. (2014)

Bottom Line: In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation.We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery.These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

No MeSH data available.


Related in: MedlinePlus

Characterization of Evo strain interaction with host cells and virulence potential.(A) Escape of C. albicans cells by piercing of macrophages (J774A.1) after different timepoints (left). Micrographs of strains after 6 h of co-incubation with J774A.1 cells (right). Intracellular C. albicans appears blue (CFW), extracellular section of the cells red (Concanavalin A, ConA). Cells of the cph1Δ/efg1Δ strain cannot escape from macrophages, while Evo cells regained this property during the evolution experiment. (B) Adhesion to and invasion of oral epithelial cells (TR-146). Adhesion values are given as percentage of adherent WT cells (left). Micrographs show filamentation of C. albicans WT and Evo strains after 6 h of incubation with TR-146 cells (right). The regained ability to filament enabled the Evo strain to invade epithelial cells. Staining was performed as described in (A). (C) Damage to macrophages and epithelial monolayers, determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation (LC = low control, medium only). WT and Evo strain, but not the cph1Δ/efg1Δ strain caused clear damage to both cell types. For piercing, adhesion, invasion and cell damage assay results are given as mean+SD of three independent experiments (*p<0.05). (D) Survival of BALB/c mice challenged intravenously (left; n = 10/strain). Nearly all mice infected with the Evo strain succumbed to the infection, while almost all animals infected with cph1Δ/efg1Δ strain survived (*p<0.05). PAS-hematoxylin-stained kidney sections from different days (d) post challenge (right) show fungal cells (arrows) either in the filamentous form (WT and Evo strain) or yeast form (cph1Δ/efg1Δ strain).
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pgen-1004824-g002: Characterization of Evo strain interaction with host cells and virulence potential.(A) Escape of C. albicans cells by piercing of macrophages (J774A.1) after different timepoints (left). Micrographs of strains after 6 h of co-incubation with J774A.1 cells (right). Intracellular C. albicans appears blue (CFW), extracellular section of the cells red (Concanavalin A, ConA). Cells of the cph1Δ/efg1Δ strain cannot escape from macrophages, while Evo cells regained this property during the evolution experiment. (B) Adhesion to and invasion of oral epithelial cells (TR-146). Adhesion values are given as percentage of adherent WT cells (left). Micrographs show filamentation of C. albicans WT and Evo strains after 6 h of incubation with TR-146 cells (right). The regained ability to filament enabled the Evo strain to invade epithelial cells. Staining was performed as described in (A). (C) Damage to macrophages and epithelial monolayers, determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation (LC = low control, medium only). WT and Evo strain, but not the cph1Δ/efg1Δ strain caused clear damage to both cell types. For piercing, adhesion, invasion and cell damage assay results are given as mean+SD of three independent experiments (*p<0.05). (D) Survival of BALB/c mice challenged intravenously (left; n = 10/strain). Nearly all mice infected with the Evo strain succumbed to the infection, while almost all animals infected with cph1Δ/efg1Δ strain survived (*p<0.05). PAS-hematoxylin-stained kidney sections from different days (d) post challenge (right) show fungal cells (arrows) either in the filamentous form (WT and Evo strain) or yeast form (cph1Δ/efg1Δ strain).

Mentions: Filamentous growth is an important contributing factor for the escape from macrophages. We therefore determined the amount of Evo cells that escaped from macrophages by piercing through their membranes after 4 h, 6 h and 8 h of co-incubation (Fig. 2A). Both Evo and wild type, but not the cph1Δ/efg1Δ double mutant, were able to escape from macrophages. However, the piercing rate of the Evo strain was significantly lower than for the wild type at all time points. After 8 h of co-incubation nearly all wild type cells had escaped from the macrophages, but only about 25% of Evo cells. The delay in filamentation and the presence of pseudohyphae in the Evo strain may explain these differences. Next, we assessed the fungus' ability to invade oral epithelial cells. Invasion requires previous adhesion, and the cph1Δ/efg1Δ strain was almost entirely unable to adhere to epithelial cells (Fig. 2B). Adhesion of the Evo strain was still reduced compared to the wild type, but significantly higher than for the double mutant (Fig. 2B). This is reflected by the invasion capacity of the Evo strain, which was significantly lower than the wild type strain, but substantially higher than the cph1Δ/efg1Δ strain. Finally, we also investigated the potential of the Evo strain to damage macrophages and epithelial cells by measuring the release of lactate dehydrogenase (LDH). After 32 hours of co-incubation, the Evo strain had damaged macrophages to the same extent as the wild type strain, and epithelial cells to a significantly higher degree than the cph1Δ/efg1Δ strain (Fig. 2C).


Microevolution of Candida albicans in macrophages restores filamentation in a nonfilamentous mutant.

Wartenberg A, Linde J, Martin R, Schreiner M, Horn F, Jacobsen ID, Je S, Wolf T, Kuchler K, Guthke R, Kurzai O, Forche A, d'Enfert C, Brunke S, Hube B - PLoS Genet. (2014)

Characterization of Evo strain interaction with host cells and virulence potential.(A) Escape of C. albicans cells by piercing of macrophages (J774A.1) after different timepoints (left). Micrographs of strains after 6 h of co-incubation with J774A.1 cells (right). Intracellular C. albicans appears blue (CFW), extracellular section of the cells red (Concanavalin A, ConA). Cells of the cph1Δ/efg1Δ strain cannot escape from macrophages, while Evo cells regained this property during the evolution experiment. (B) Adhesion to and invasion of oral epithelial cells (TR-146). Adhesion values are given as percentage of adherent WT cells (left). Micrographs show filamentation of C. albicans WT and Evo strains after 6 h of incubation with TR-146 cells (right). The regained ability to filament enabled the Evo strain to invade epithelial cells. Staining was performed as described in (A). (C) Damage to macrophages and epithelial monolayers, determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation (LC = low control, medium only). WT and Evo strain, but not the cph1Δ/efg1Δ strain caused clear damage to both cell types. For piercing, adhesion, invasion and cell damage assay results are given as mean+SD of three independent experiments (*p<0.05). (D) Survival of BALB/c mice challenged intravenously (left; n = 10/strain). Nearly all mice infected with the Evo strain succumbed to the infection, while almost all animals infected with cph1Δ/efg1Δ strain survived (*p<0.05). PAS-hematoxylin-stained kidney sections from different days (d) post challenge (right) show fungal cells (arrows) either in the filamentous form (WT and Evo strain) or yeast form (cph1Δ/efg1Δ strain).
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Related In: Results  -  Collection

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pgen-1004824-g002: Characterization of Evo strain interaction with host cells and virulence potential.(A) Escape of C. albicans cells by piercing of macrophages (J774A.1) after different timepoints (left). Micrographs of strains after 6 h of co-incubation with J774A.1 cells (right). Intracellular C. albicans appears blue (CFW), extracellular section of the cells red (Concanavalin A, ConA). Cells of the cph1Δ/efg1Δ strain cannot escape from macrophages, while Evo cells regained this property during the evolution experiment. (B) Adhesion to and invasion of oral epithelial cells (TR-146). Adhesion values are given as percentage of adherent WT cells (left). Micrographs show filamentation of C. albicans WT and Evo strains after 6 h of incubation with TR-146 cells (right). The regained ability to filament enabled the Evo strain to invade epithelial cells. Staining was performed as described in (A). (C) Damage to macrophages and epithelial monolayers, determined by lactate dehydrogenase (LDH) assay after 32 h of co-incubation (LC = low control, medium only). WT and Evo strain, but not the cph1Δ/efg1Δ strain caused clear damage to both cell types. For piercing, adhesion, invasion and cell damage assay results are given as mean+SD of three independent experiments (*p<0.05). (D) Survival of BALB/c mice challenged intravenously (left; n = 10/strain). Nearly all mice infected with the Evo strain succumbed to the infection, while almost all animals infected with cph1Δ/efg1Δ strain survived (*p<0.05). PAS-hematoxylin-stained kidney sections from different days (d) post challenge (right) show fungal cells (arrows) either in the filamentous form (WT and Evo strain) or yeast form (cph1Δ/efg1Δ strain).
Mentions: Filamentous growth is an important contributing factor for the escape from macrophages. We therefore determined the amount of Evo cells that escaped from macrophages by piercing through their membranes after 4 h, 6 h and 8 h of co-incubation (Fig. 2A). Both Evo and wild type, but not the cph1Δ/efg1Δ double mutant, were able to escape from macrophages. However, the piercing rate of the Evo strain was significantly lower than for the wild type at all time points. After 8 h of co-incubation nearly all wild type cells had escaped from the macrophages, but only about 25% of Evo cells. The delay in filamentation and the presence of pseudohyphae in the Evo strain may explain these differences. Next, we assessed the fungus' ability to invade oral epithelial cells. Invasion requires previous adhesion, and the cph1Δ/efg1Δ strain was almost entirely unable to adhere to epithelial cells (Fig. 2B). Adhesion of the Evo strain was still reduced compared to the wild type, but significantly higher than for the double mutant (Fig. 2B). This is reflected by the invasion capacity of the Evo strain, which was significantly lower than the wild type strain, but substantially higher than the cph1Δ/efg1Δ strain. Finally, we also investigated the potential of the Evo strain to damage macrophages and epithelial cells by measuring the release of lactate dehydrogenase (LDH). After 32 hours of co-incubation, the Evo strain had damaged macrophages to the same extent as the wild type strain, and epithelial cells to a significantly higher degree than the cph1Δ/efg1Δ strain (Fig. 2C).

Bottom Line: In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation.We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery.These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbial Pathogenicity Mechanisms, Leibniz Institute for Natural Product Research and Infection Biology - Hans Knoell Institute Jena (HKI), Jena, Germany.

ABSTRACT
Following antifungal treatment, Candida albicans, and other human pathogenic fungi can undergo microevolution, which leads to the emergence of drug resistance. However, the capacity for microevolutionary adaptation of fungi goes beyond the development of resistance against antifungals. Here we used an experimental microevolution approach to show that one of the central pathogenicity mechanisms of C. albicans, the yeast-to-hyphae transition, can be subject to experimental evolution. The C. albicans cph1Δ/efg1Δ mutant is nonfilamentous, as central signaling pathways linking environmental cues to hyphal formation are disrupted. We subjected this mutant to constant selection pressure in the hostile environment of the macrophage phagosome. In a comparatively short time-frame, the mutant evolved the ability to escape macrophages by filamentation. In addition, the evolved mutant exhibited hyper-virulence in a murine infection model and an altered cell wall composition compared to the cph1Δ/efg1Δ strain. Moreover, the transcriptional regulation of hyphae-associated, and other pathogenicity-related genes became re-responsive to environmental cues in the evolved strain. We went on to identify the causative missense mutation via whole genome- and transcriptome-sequencing: a single nucleotide exchange took place within SSN3 that encodes a component of the Cdk8 module of the Mediator complex, which links transcription factors with the general transcription machinery. This mutation was responsible for the reconnection of the hyphal growth program with environmental signals in the evolved strain and was sufficient to bypass Efg1/Cph1-dependent filamentation. These data demonstrate that even central transcriptional networks can be remodeled very quickly under appropriate selection pressure.

No MeSH data available.


Related in: MedlinePlus